scholarly journals Amino Acid 226 in the Hemagglutinin of H4N6 Influenza Virus Determines Binding Affinity for α2,6-Linked Sialic Acid and Infectivity Levels in Primary Swine and Human Respiratory Epithelial Cells

2008 ◽  
Vol 82 (16) ◽  
pp. 8204-8209 ◽  
Author(s):  
Allen C. Bateman ◽  
Marc G. Busch ◽  
Alexander I. Karasin ◽  
Nicolai Bovin ◽  
Christopher W. Olsen
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
Sialic Acid ◽  
Amino Acid ◽  
Epithelial Cells ◽  
Influenza Viruses ◽  
Respiratory Epithelial Cells ◽  
Relative Binding ◽  
Human Respiratory Epithelial Cells ◽  
Respiratory Epithelial

ABSTRACT Avian lineage H4N6 influenza viruses previously isolated from pigs differ at hemagglutinin amino acids 226 and 228 from H4 subtype viruses isolated from birds. Using a parental H4N6 swine isolate and hemagglutinin mutant viruses (at residues 226 and/or 228), we determined that viruses which contain L226 had a higher affinity for sialic acid α2,6 galactose (SAα2,6Gal) and a higher infectivity level for primary swine and human respiratory epithelial cells, whereas viruses which contain Q226 had lower SAα2,6Gal affinity and lower infectivity levels for both types of cells. Using specific neuraminidases, we found that irrespective of their relative binding preferences, all of the influenza viruses examined utilized SAα2,6Gal to infect swine and human cells.

scholarly journals Primary Swine Respiratory Epithelial Cell Lines for the Efficient Isolation and Propagation of Influenza A Viruses

2020 ◽  
Vol 94 (24) ◽  
Author(s):  
Victoria Meliopoulos ◽  
Sean Cherry ◽  
Nicholas Wohlgemuth ◽  
Rebekah Honce ◽  
Karen Barnard ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Influenza A ◽  
Vaccine Development ◽  
Mdck Cells ◽  
Influenza Viruses ◽  
Clinical Samples ◽  
Respiratory Epithelial Cells ◽  
Respiratory Epithelial

ABSTRACT Influenza virus isolation from clinical samples is critical for the identification and characterization of circulating and emerging viruses. Yet efficient isolation can be difficult. In these studies, we isolated primary swine nasal and tracheal respiratory epithelial cells and immortalized swine nasal epithelial cells (siNEC) and tracheal epithelial cells (siTEC) that retained the abilities to form tight junctions and cilia and to differentiate at the air-liquid interface like primary cells. Critically, both human and swine influenza viruses replicated in the immortalized cells, which generally yielded higher-titer viral isolates from human and swine nasal swabs, supported the replication of isolates that failed to grow in Madin-Darby canine kidney (MDCK) cells, and resulted in fewer dominating mutations during viral passaging than MDCK cells. IMPORTANCE Robust in vitro culture systems for influenza virus are critically needed. MDCK cells, the most widely used cell line for influenza isolation and propagation, do not adequately model the respiratory tract. Therefore, many clinical isolates, both animal and human, are unable to be isolated and characterized, limiting our understanding of currently circulating influenza viruses. We have developed immortalized swine respiratory epithelial cells that retain the ability to differentiate and can support influenza replication and isolation. These cell lines can be used as additional tools to enhance influenza research and vaccine development.

Identification of amino acids in the HA of H3 influenza viruses that determine infectivity levels in primary swine respiratory epithelial cells

2008 ◽  
Vol 133 (2) ◽  
pp. 269-279 ◽  
Author(s):  
Marc G. Busch ◽  
Allen C. Bateman ◽  
Gabriele A. Landolt ◽  
Alexander I. Karasin ◽  
Rebecca A. Brockman-Schneider ◽  
...  
Keyword(s):  
Amino Acids ◽  
Epithelial Cells ◽  
Influenza Viruses ◽  
Respiratory Epithelial Cells ◽  
Respiratory Epithelial

scholarly journals Functional Analysis of PA Binding by Influenza A Virus PB1: Effects on Polymerase Activity and Viral Infectivity

2001 ◽  
Vol 75 (17) ◽  
pp. 8127-8136 ◽  
Author(s):  
Daniel R. Perez ◽  
Ruben O. Donis
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
Amino Acid ◽  
Viral Replication ◽  
Influenza A Virus ◽  
Influenza A ◽  
Amino Acid Sequences ◽  
Influenza Viruses ◽  
Virus Growth ◽  
Transcription And Replication

ABSTRACT Influenza A virus expresses three viral polymerase (P) subunits—PB1, PB2, and PA—all of which are essential for RNA and viral replication. The functions of P proteins in transcription and replication have been partially elucidated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Although it is conceivable that heterotrimer subunit interactions may allow a more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PB1 and PB2 as well as between PB1 and PA. Previous studies have shown that the N-terminal 48 amino acids of PB1, termed domain α, contain the residues required for binding PA. We report here the refined mapping of the amino acid sequences within this small region of PB1 that are indispensable for binding PA by deletion mutagenesis of PB1 in a two-hybrid assay. Subsequently, we used site-directed mutagenesis to identify the critical amino acid residues of PB1 for interaction with PA in vivo. The first 12 amino acids of PB1 were found to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain α. The role of the minimal PB1 domain α in influenza virus gene expression and genome replication was subsequently analyzed by evaluating the activity of a set of PB1 mutants in a model reporter minigenome system. A strong correlation was observed between a functional PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse genetics system. Interestingly, mutations that rendered PB1 unable to bind PA were either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PB1 in binding PA, P activity, and virus growth.

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